JP6803334B2 - Aggregate-Method for detecting aggregated form of forming polypeptide - Google Patents

Description

This patent application claims priority over the Republic of Korea Patent Application No. 10-2014-017608 filed with the Republic of Korea Patent Office on December 02, 2014, and the disclosure items of the patent application are described in the present specification. Captured as a reference.

The present invention relates to a method or kit for detecting an aggregated form of a raw sample (biosample) -an aggregated form of a forming polypeptide.

First, the polypeptides that make up a protein may form a multimer to form a functional protein, but in the normal state, they exist as monomers and are in an abnormal state (for example, misfolding). In many cases, proteins are formed and aggregated to induce diseases (Non-Patent Documents 1 and 2).

For example, diseases or diseases associated with abnormal protein aggregation or misfolding include Alzheimer's disease, Kreuzfeld-Jakob's disease, Spongiform encephalopasias, Parkinson's disease, Huntington's disease, muscle atrophic lateral sclerosis. (Amyotropic lateral disease), Serpin deficiency, pulmonary emphysema, cirrhosis, type II diabetes, primary systemic amyloidosis, secondary systemic dementia, frontal dementia, frontal dementia, frontal dementia ), Senile systemic amyloidosis, familial amyloid polyneuropathy, hereditary cerebral amyloid angiopathy and hemodialysis-related amyloidosis.

In measuring the presence or absence of these diseases or the degree of progression, the amount of antigen in the sample is very small, or the size of the antigen is so small that it is difficult to measure, or the amount of antigen in the body. When the amount of antigen in the sample is not proportional, for example, Aβ (amyloid-beta) involved in Alzheimer's disease is also known to have a higher level of Aβ oligomer in abnormal subjects than in normal subjects. However, if it is difficult to detect the amount of Aβ oligomer in the blood sample, or if Aβ oligomer is atypically present in the blood sample, diagnosis may be difficult.

In addition, the antigen to be measured may be too small or too small to easily diagnose the disease via sandwich ELISA.

Therefore, the present inventors recognized the necessity of developing a method for detecting agglutinated-forming polypeptides by maximizing the difference in diagnostic signals between patients and normal humans.

A large number of articles and patent documents are referred to throughout the specification, and their citations are displayed. The disclosed contents of the cited treatises and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are explained more clearly.

Massimo Stefani, et al. , J. Mol. Med. 81: 678-699 (2003) Radford SE, et al. , Cell. 97: 291-298 (1999)

We have worked diligently to develop new methods for detecting agglutinated forms of agglutinating-forming polypeptides, and as a result, a defensive system (clearing system) that suppresses the formation of agglutinating forms of polypeptides. Utilizing the difference in hydrophobic interaction, we have developed a method for detecting agglutinated forms of agglutinated-formed polypeptides by maximizing the difference in diagnostic signals between patients and normal humans.

Therefore, an object of the present invention is to provide a method for detecting an aggregated form of a raw sample (biosample) -an aggregated form of a forming polypeptide.

Another object of the present invention is to provide a kit for detecting the aggregated form of a raw sample (biosample) -the aggregated form of a forming polypeptide.

Other objects and advantages of the present invention will be further clarified by the detailed description of the invention, the scope of claims and the drawings.

According to the uniformity of the present invention, the present invention provides a method for detecting the aggregated form of a raw sample-aggregated form of a forming polypeptide, including the following steps: (a) the raw sample to be analyzed (i). Monomeric or multimer form of aggregated-forming polypeptide, (iii) hydrophobic deletion of aggregate polypeptide of said aggregated-formed polypeptide, or (iii) said aggregated-formed polypeptide. The step of spiked the monomeric or multimer form of the peptide with the hydrophobic deletion of the aggregate-forming polypeptide, and (b) the result of the step (a) being incubated to the aggregate- The step of additionally forming an aggregated form of the forming polypeptide (where the incubation is carried out long enough for the spiked (i), (ii) or (iii) to be multimerized by the raw sample). , (C) The result of the step (b) is brought into contact with a binder-label in which a signal generating label is bound to a binder that binds to the aggregate-forming polypeptide, and (d). A step of detecting a signal generated from a binder-label bound to the aggregated form of the aggregated polypeptide.

The present invention maximizes the difference in diagnostic signals between patients and normal humans by utilizing the difference in the defense system (clearing system) or hydrophobic interaction that suppresses the formation of aggregates of polypeptides. The present invention relates to a method for detecting an aggregated form of an aggregated-formed polypeptide.

The features and advantages of the present invention can be summarized as follows:
(A) The present invention provides a method or a kit for detecting an aggregated form of a raw sample (biosample) -an aggregated form of a forming polypeptide.
(B) In the method of the present invention, the amount of aggregated form (antigen) of the aggregated form-forming polypeptide to be measured in the raw sample is very small, or the size thereof is very small, and the measurement can be performed. Difficult or when the amount of aggregated-formed polypeptide (antigen) in the human body is not proportional to the amount of aggregated-formed polypeptide (antigen) in the raw sample Differences in defensive systems that suppress agglutination and / or hydrophobic interactions were used to maximize the difference in diagnostic signals between patients and normal humans.
(C) The present invention can be carried out in a convenient and rapid manner, which allows automation of the method of detecting the aggregated form of the biosample-the aggregated form of the forming polypeptide.

FIG. 1a shows rec. After Aβ1-42 spikes, changes in Aβ oligomers with a 4-day incubation time are shown. FIG. 1b shows rec. After Aβ1-42 spikes, changes in Aβ oligomers with 6 days incubation time are shown. FIG. 1c shows rec. After Aβ1-42 spikes, changes in Aβ oligomers with incubation time of 2, 3, 4, and 5 days are shown. FIG. 1d shows rec. After Aβ1-42 spike, 5 days incubation, rec. After incubation for 0 days and 5 days without the addition of Aβ1-42, changes in Aβ oligomers are shown. FIG. 2a shows rec. After spiked with Aβ9-42 biotin and incubated for 6 days, changes in Aβ oligomers are shown. FIG. 2b shows rec. Binding to Aβ. After spiked Aβ9-42, after incubation for 2 days, 3 days and 4 days, changes in Aβ oligomers are shown. FIG. 3 shows changes in α-synuclein oligomers over time for 0 days, 2 days, 4 days, and 6 days of incubation after spiked recombinant α-synuclein.

As used herein, the term'aggregate-forming polypeptide' means a polypeptide capable of forming a multimer type (oligomeric type) or forming an aggregated form by hydrophobic interaction with a monomer. To do. In particular, the following structural changes induce various diseases. For example, Alzheimer's disease, Kreuzfeld-Jakob's disease, Spongiform encephalopasias, Parkinson's disease, Huntington's disease, amyloid lateral sclerosis, Serpine deficiency (Serpine) , Cirrhosis, type II diabetes, primary systemic amyloidosis, secondary systemic amyloidosis, fronto-temporal dementia, senile systemic amyloidosis, familial amyloid polyneuropathy, family amyloidosis ), Hereditary cerebral amyloid angiopathy and hemodialysis-related amyloidosis.

In general, the non-aggregated form of the aggregated-forming polypeptide is normal and the aggregated form induces diseases, especially degenerative neurological diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease or Parkinson's disease.

According to one embodiment of the invention, the raw sample that multimerizes the spiked (i), (ii) or (iii) is a human having a disease associated with the multimer form of the aggregate-forming polypeptide. A raw sample, more preferably a signal generated utilizing a human raw sample with a disease associated with the multimer form of aggregate-forming polypeptide, for an incubation time sufficient to be multimerized by the raw sample. It is sufficient time to make the signal 1.5 to 20 times larger than the signal generated using a normal human raw sample.

Hereinafter, the method of the present invention for detecting the aggregated form of a raw sample (biosample) -the aggregated form of the forming polypeptide will be described in detail step by step as follows.

(A) Spike Stage First, the method of the present invention comprises (i) the monomer type or multimer type (oligomer type) of the aggregate-formed polypeptide, and (ii) the aggregate-formation of the raw sample to be analyzed. Hydrophobic deletion of aggregate-forming polypeptide, or (iii) monomeric or multimer (oligomeric) or multimer (oligomer) forms of the aggregated-forming polypeptide and hydrophobicity of the aggregated-forming polypeptide. It includes a step of spiking with a sex deletion substance.

As used herein, the term'biosample' means a sample derived from an organism to be analyzed. Said raw sample refers to a cell, tissue, or biofluid of a biological source, or other medium that can be analyzed by the present invention, which is a sample taken from a human, a sample taken from an animal, a human or an animal. Includes samples taken from food for. Preferably, the raw sample to be analyzed is blood, serum, plasma, lymph, milk, urine, stool, tears, saliva, semen, brain extract (eg, brain homologous fluid), spinal fluid (SCF), wormdrops, spleen. , And a body fluid sample containing tonsillar tissue extract. More preferably, the raw sample is blood, most preferably plasma.

According to other embodiments of the present invention, the aggregated-forming polypeptides are involved in Aβ peptides and tau proteins involved in Alzheimer's disease, prions involved in Kreuzfeld-Jakob disease and spongeform brain disease, and Parkinson's disease. α-Synuclein, Ig light chain involved in primary systemic amyloidosis, serum amyloid A involved in secondary systemic amyloidosis, tau protein involved in frontal temporal lobar dementia, transsiletin involved in senile systemic amyloidosis , Transsiletin involved in familial amyloid polyneuropathy, Cystatin C involved in hereditary cerebral amyloidosis, β2-microglobulin involved in hemodialysis-related amyloidosis, Huntington's disease involved in Huntington's disease, muscle atrophic lateral cord Includes superoxide dismutase involved in sclerosis, selpin deficiency, pulmonary emphysema, and celpin involved in liver cirrhosis, and amyloid involved in type II diabetes. More preferably, the aggregated-forming polypeptide is an Aβ peptide or tau protein involved in Alzheimer's disease, or an α-synuclein involved in Parkinson's disease, and most preferably an Aβ peptide or α-synuclein.

As used herein, the term'spiking'refers to the raw sample to be analyzed as a monomeric (or multimer) form of the aggregated-formed polypeptide and / or a hydrophobic deletion of the aggregated-formed polypeptide. Means the process of adding or mixing after addition.

As used herein, the term'multimer'is formed by combining two or more monomers, which also includes oligomers.

According to the present invention, (i) a defense system that suppresses the formation of the aggregated form of the aggregated-formed polypeptide when the monomer type or multimer type of the aggregated-formed polypeptide is spiked on the raw sample to be analyzed (i). Utilizing the difference in the clearing system, the difference between the diagnostic signal of the patient and the normal human is maximized, that is, the patient's raw sample has a low degree of defense system, and the aggregated-formed polypeptide. While the aggregate formation of peptides is promoted, in normal human raw samples, the degree of defense system (clearing system) is high, the aggregate formation of aggregate-forming polypeptides is reduced, and the difference in diagnostic signals ( Differation) is maximized.

According to yet another embodiment of the present invention, the monomer type of the aggregated-formed polypeptide is an Aβ peptide consisting of the amino acid sequence of SEQ ID NO: 1 or an α-synuclein consisting of the amino acid sequence of SEQ ID NO: 2.

According to the present invention, when (ii) a hydrophobic deletion of the aggregate-forming polypeptide is spiked on a raw sample to be analyzed, the hydrophobicity (hydrophic) is observed. Hydrophobic interaction between a hydrophobic deletion of an aggregate-forming polypeptide containing an amino acid residue having () and a monomer-type or multimer type (oligoform type) of the aggregate-formed polypeptide present in a raw sample. Utilize the difference in interaction) to maximize the difference in diagnostic signals between patients and normal humans, that is, the monomeric or multimer type (oligoform type) of aggregate-forming polypeptides present in the patient's raw sample. Hydrophobic deletions of aggregated-formed polypeptides form large amounts of aggregated forms due to hydrophobic interactions, but monomeric and aggregated-formed aggregated-formed polypeptides present in normal human raw samples. Hydrophobic deletions of polypeptides form aggregates through hydrophobic interactions, but are less than in patients, maximizing the difference in diagnostic signals between patients and normal humans. ..

As used herein, the term'hydrophobic delegated divergent of aggregate-forming polypeptide' refers to the monomeric or multimer (oligoform) and hydrophobic forms of aggregated-forming polypeptides. It is a derivative in which amino acid residues are deleted so as to contain a large number of hydrophobic amino acid residues from the amino acid sequence of the aggregated-formed polypeptide so that the aggregated form can be formed by sexual interaction.

Hydrophobic deletions of aggregated-formed polypeptides are length (molecular weight) and / or hydrophobic amino acids due to hydrophobic interactions with the monomeric or multimer (oligoform) forms of aggregated-formed polypeptides. The residue can be selected in consideration of the residue, and preferably, the hydrophobic deletion of the aggregated-forming polypeptide (hydrophobic detached divergent of aggregate-forming carrying peptide) is the 37th amino acid of the amino acid sequence of SEQ ID NO: 1. It is an Aβ _delete peptide containing residues to the 42nd amino acid residue, and more preferably, the Aβ _delete peptide is a peptide containing the 29th amino acid residue to the 42nd amino acid residue of the amino acid sequence of SEQ ID NO: 1. More preferably, the Aβ _delete peptide is a peptide containing the 17th to 42nd amino acid residues of the amino acid sequence of SEQ ID NO: 1, and most preferably, the Aβ _delete peptide is the amino acid of SEQ ID NO: 1. It is a peptide containing the 9th to 42nd amino acid residues of the sequence.

According to the present invention, when (iii) a monomer type or multimer type of the aggregated-formed polypeptide and a hydrophobic deletion product of the aggregated-formed polypeptide are spiked in the raw sample to be analyzed. To take advantage of both the effects achieved by spikes the monomeric or multimer form of (i) the aggregated-forming polypeptide and the hydrophobic deletion of (ii) the aggregated-formed polypeptide, respectively. That is, the difference in the diagnostic signal between the patient and the normal human is maximized by utilizing the difference in the defense system (clearing system) and the hydrophobic interaction (hydrophobic intervention) that suppress the aggregation formation of the polypeptide. To do.

According to another embodiment of the present invention, a buffer solution is further added to the product of the step (a). More preferably, the buffer is added 3 to 15 times (v / v) with respect to the raw sample, more preferably 5 to 13 times (v / v), and even more preferably. It is added 7 to 11 times (v / v), most preferably 8 to 10 times (v / v).

As the buffer solution used in the present invention, various buffer solutions known in the art can be used, and the buffer solution is preferably a nonionic surfactant-containing phosphate buffer solution.

As the nonionic surfactant contained in the phosphate buffer used in the present invention, various nonionic surfactants known in the art can be utilized, preferably alkyl alkoxylated. Includes ethers, alkoxylated alkyl esters, alkyl polyglycosides, polyglyceryl esters, polysorbates and sugar esters. More preferably, Tween-20 or Triton X-100 is used, and most preferably Tween-20 is used.

(B) Step of additionally forming the aggregated form of the aggregated-formed polypeptide The method of the present invention is (b) the aggregated form of the aggregated-formed polypeptide by incubating the result of the step (a). Including the step of forming an additional.

One of the greatest features of the present invention is that the amount of the aggregated form (antigen) of the aggregated-formed polypeptide to be measured in the raw sample is very small or the size is very small. If it is difficult, or if the amount of aggregated form (antigen) of the aggregated-formed polypeptide in the human body is not proportional to the amount of aggregated form (antigen) of the aggregated form-formed polypeptide in the raw sample, the above (I) Monomer or multimer type of the aggregated-forming polypeptide, (ii) Hydrophobic delegated divergent of aggressive-formed peptide, or (ii) said. The monomer type or multimer type of the aggregated-formed polypeptide and the hydrophobic deletion product of the aggregated-formed polypeptide are spiked into a raw sample to add the aggregated form of the aggregated-formed polypeptide. It means that the presence or absence of a disease or a disease and the degree of progression can be measured by forming the peptide.

According to another embodiment of the invention, the additional formation of the aggregated form of the aggregated polypeptide of said step (b) involves incubating the product of said step (a) at a temperature of 1-50 ° C. It is performed by incubation), more preferably by incubation at a temperature of 25 to 50 ° C, even more preferably by a temperature of 25 to 45 ° C, and even more preferably by a temperature of 25 to 40 ° C. Most preferably, the temperature is 25 to 38 ° C.

In the present invention, the incubation of the step (b) is carried out for a sufficient time so that the spiked (i), (ii) or (iii) can be multimerized with the raw sample, more preferably with the raw sample. The incubation time sufficient for multimerization is 1 from the signal generated using a human raw sample with a disease involving the multimer form of the aggregate-forming polypeptide from the signal generated using a normal human raw sample. It is enough time to make it 5 to 20 times larger.

According to yet another embodiment of the present invention, the signal generated using the raw sample of the patient is carried out for a sufficient time to be 1.5 to 20 times larger than the signal generated using the raw sample of a normal person. The additional formation of the aggregated form of the aggregated polypeptide of said step (b) is preferably carried out by incubating the result of said step (a) for 1 to 12 days. Is carried out for 30 hours (hr) to 10 days, more preferably carried out for 2 to 8 days, further preferably carried out for 2 to 6 days, further preferably carried out for 3 to 6 days, and most preferably. Is carried out for 4 to 6 days, more preferably 5 to 6 days.

As used herein, the term'incubation' means to stand or shake a raw sample to be analyzed at a predetermined temperature for a period of time, preferably when shaken. It means to do a mild shaking.

Another of the greatest features of the present invention is the monomeric form of the spiked aggregate-forming polypeptide present in the raw sample by standing (ie, incubating) the raw sample at a predetermined temperature for a period of time. Differences in diagnostic signals between patients and normal humans by allowing the hydrophobic deletion of the aggregated-forming polypeptide and / or the aggregated-formed polypeptide to be well aggregated with each other. This is the maximization of (diflation).

(C) Binder-labeled contact with the result of step (b) that binds to the aggregate of the aggregate-forming polypeptide The method of the invention comprises (c) the result of step (b). It goes through a step of contacting the binder-label with the signaling label bound to the binder that binds to the aggregate-forming polypeptide.

In the present invention, the binding agent that binds to the aggregated form of the aggregated-forming polypeptide is antibody, peptide aptamer, AdNectin, affinity (US Pat. No. 5,831,012), Avimmer (Silverman, J. Mol. et al, Nature Biotechnology 23 (12): 1556 (2005)), or Kunitz drug (Arnoux B et al., Acta Crystallogr. D Biol. Crystallogr. 58 (Pt7), Axtallogr. 58 (Pt7), 12524 (200)) Current option in drug discovery & development 9 (2): 2618 (2006)).

In the present invention, the signal-generating label bound to the binding agent that binds to the aggregated form of the aggregated-forming polypeptide is a compound label (eg, biotin), an enzyme label (eg, alkaline phosphatase, peroxydase, β- Galactosidase and β-glucosidase), radiolabels (eg, I ¹²⁵ , and C ¹⁴ ), fluorescent labels (eg, fluorescein), luminescent labels, chemical luminescent labels, and FRET (fluorescence resonance energy transfer) labels. However, but is not limited to these.

(D) Detection of Signals Generated from Binders-labels Bindered to Aggregate Form-Forming Polypeptides Finally, the method of the invention is (d) to aggregated forms of the aggregated-forming polypeptide. Includes the step of detecting the signal emanating from the bound binder-label.

Detection of the signal generated from the agglutinating-binding agent-label bound to the agglutinating polypeptide can be carried out by various methods known in the art, for example, involving an antigen-antibody reaction. It can be performed using immunoassay.

According to yet another embodiment of the present invention, the steps (c) and (c) are carried out in a manner that includes the following steps: (c-1) The aggregated form that captures the aggregated form. -The step of contacting the capture antibody that recognizes the epitope on the forming polypeptide with the product of the step (b), and (c-2) the detection antibody that recognizes the epitope on the aggregated-forming polypeptide. A step of contacting the captured aggregated form and a step of detecting the (c-3) aggregated form-detection antibody complex.

Such a detection method utilizes two forms of antibody, namely a captive antibody and a detection antibody. As used herein, the term'capture antibody' means an antibody capable of binding to an aggregate-forming polypeptide to be detected from a raw sample. The term'detection antibody' means an antibody capable of binding to an aggregate-forming polypeptide captured by the capture antibody. 'Antibody' means an immunoglobulin protein that can bind to an antigen. Antibodies utilized herein include antibody fragments (eg, F (ab') 2, Fab', Fab, Fv) as well as whole antibodies capable of binding to the epitope, antigen or antigen fragment to be detected. ..

The detection method utilizes a set of a capture antibody and a detection antibody that specifically recognize an epitope on an aggregated-forming polypeptide, and the epitope that the capture antibody and the detection antibody specifically recognize. Are identical or overlap each other.

The term'overlapped with', used with reference to epitopes for captured and detected antibodies, includes epitopes containing fully or partially overlapped amino acid sequences. For example, the epitopes for 6E10 and WO2 antibodies have amino acid sequences consisting of amino acids 3-8 and 4-10, respectively, of the human Aβ peptide sequences, and the epitopes for 6E10 and FF51 antibodies have amino acids, respectively, of the human Aβ peptide sequences. It has an amino acid sequence consisting of 3-8 and 1-4, and the epitope for the 1E11 and WO2 antibodies has an amino acid sequence consisting of amino acids 1-8 and 4-10, respectively, of the human Aβ peptide sequence, 1E11 and FF51. The epitope for the antibody has an amino acid sequence consisting of amino acids 1-8 and 1-4, respectively, of the human Aβ peptide sequence. These epitopes can be described as completely overlapping epitopes.

The epitope for the 3B6 and 3B6 biotin antibodies is a sequence consisting of 119-140 of the α-synuclein protein sequence.

According to another embodiment of the invention, when expressed with reference to the human Aβ peptide sequence, the epitope has an amino acid sequence consisting of amino acids 1-8, 3-8, 1-4 or 4-10. When expressed with reference to the α-synuclein protein sequence, the epitope has an amino acid sequence consisting of amino acids 119-140.

According to yet another embodiment of the present invention, the epitope recognized by the capture antibody is a sequence that is not repeated in the aggregate-forming polypeptide, and the epitope recognized by the detection antibody is the aggregate-forming poly. A sequence that is not repeated with peptides. According to the detection method of the present invention, the aggregated-forming polypeptide bound to the capture antibody cannot bind further to the detection antibody, because there is no additional epitope recognized by the detection antibody. Is.

According to another embodiment of the present invention, the capture antibody and the detection antibody are identical to each other. That is, it is preferable that the epitopes specifically bound to the capture antibody and the detection antibody are the same.

According to yet another embodiment of the present invention, the captured antibody is bound to a solid substrate. Known materials in this form include hydrocarbon polymers such as polystyrene and polypropylene, glass, metals, and gels. The solid substrate can be in the form of dipsticks, microtiter plates, particles (eg beads), affinity columns and immunoglobulin membranes (eg polyvinylidene fluoride membranes) (see US Pat. No. 5,143,825). , Nos. 5,374,530, 4,908,305 and 5,498,551).

According to another embodiment of the invention, the detection antibody has a label that produces a detectable signal. The labels include compound labels (eg, biotin), enzyme labels (eg, alkaline phosphatase, peroxydase, β-galactosidase and β-glucosidase), radiolabels (eg, I ¹²⁵ , and C ¹⁴ ), fluorescent labels (eg, I ¹²⁵ , and C ¹⁴ ). For example, fluorescein), luminescent labels, chemical luminescent labels, and FRET (fluorescence resonance energy transfer) labels are included, but are not limited to. Various covers and methods for labeling antibodies are known in the art (Harrow and Lane, eds. Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring. ..

In the present invention, an antibody capable of binding to an aggregate-forming polypeptide is fused (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), a recombinant DNA method (US Pat. No. 4,816,56). No.) or conventional phage antibody libraries (Clackson et al, Nature, 352: 624-628 (1991); and Marks et al, J. Mol. Biol. 222: 58, 1-597 (1991)). According to the technique, it can be prepared by utilizing an epitope conventionally described as an immunogen. Common methods for the production of said antibodies are described in Harlow, E. et al. and Lane, D.I. , Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1988; Zola, H. et al. , Monoclonal Antibodies: A Manual of Technologies, CRC Press, Inc. , Boca Raton, Florida, 1984; and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991.

Preparation of a hybridoma cell line for the production of a monoclonal antibody is carried out by fusion of an immortal cell line with antibody-producing lymphocytes. The production of the monoclonal antibody can be carried out using a technique known in the art. A polyclonal antibody can be produced by injecting the above-mentioned antigen into a suitable animal, collecting an antiserum containing the antibody, and separating the antibody by a known affinity technique.

Detection of the aggregate-detection antibody complex can be performed by a variety of methods known in the art. The formation of the aggregate-detection antibody complex indicates the presence of the aggregate in the raw sample. The steps are performed according to conventional methods, for example, Enzyme Immunoassay, E.I. T. Maggio, ed. , CRC Press, Boca Raton, Florida, 1980, and Harlow and Lane, eds. Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.K. As described in Y, it can be performed quantitatively or qualitatively with a variety of detectable label / substrate pairs.

When the detection antibody is labeled with alkaline phosphatase, bromochloroindoyl phosphatase (BCIP), nitroblue tetrazolium (NBT) and ECF can be used as substrates for the color development reaction. When labeling with horseradish peroxidase, as substrates, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacrydinium nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-Acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine), ECL (enhanced chemiluminescence) and ABTS (2,2-azine-di [3-ethylbenzothiazolin) Sulfonate]) and the like are used.

In this way, the signal generated using a human raw sample with a disease involving the multimer form of the aggregated-forming polypeptide is more than the signal generated using a normal human raw sample. It can be increased by 5 to 20 times, more preferably 1.5 to 10 times, and more preferably 1.6 to 10 times.

According to another aspect of the invention, the invention provides a kit for detecting an aggregated-formed polypeptide of a raw sample (biosample) including: (i) said aggregated-formed poly. Monomeric or multimer form of the peptide, (ii) hydrophobic deleted divergent of aggregate-forming carrying peptide, or (iii) monomer type of the aggregate-forming polypeptide or Hydromeric deletions of the multimer type and the aggregated-forming polypeptide.

The kit of the present invention utilizes the above-mentioned method of detecting the aggregated form of the raw sample (biosample) of the present invention-the aggregated form of the forming polypeptide, and the common content thereof is an excess of the present specification. The description is omitted to avoid complexity.

According to another embodiment of the invention, the kit further comprises a capture antibody that recognizes an epitope on an aggregate-forming polypeptide and a detection antibody that recognizes the epitope recognized by the capture antibody.

Hereinafter, the present invention will be described in more detail through Examples, but these Examples are for more specific explanation of the present invention, and the scope of the present invention is not limited to these Examples. It will be obvious to those who have ordinary knowledge in the technical field to which the present invention belongs.

Example 1: Preparation of Experimental Materials Coating buffer (Carbonate-Bicarbonate Buffer), PBST, TBST and PBS were purchased from Sigma. Block Ace was purchased from Bio-rad. Buffer A was produced by diluting Block Ace to 0.4% in TBST. The blocking buffer is described in D.I. Manufactured so that 1% Block Ace was diluted with W. The 6E10 antibody was purchased from BioLegend. The 3B6 antibody was purchased from Novus Biologicals. For the 3B6-biotin antibody, an antibody purchased from Novus Biologicals was biotinylated by Peoplebio. Streptavidin-HRP was purchased from Thermo Scientific. HBR1 was purchased from Scantibodies Laboratory. FF51-HRP was purchased from The Hlab Co., Ltd. Recombinant Aβ1-42 was purchased from BioLegend. Recombinant Aβ9-42 biotin was purchased from Anaspec. Recombinant Aβ9-42 was purchased from Anaspec. Recombinant α-synuclein was purchased from Milipore. Plasma samples were obtained from Seoul National University Hospital and Central University Hospital. The ECL solution was purchased from Rockland. The plate was purchased from Nunc. The epitope for the 6E10 and FF51 antibodies has an amino acid sequence consisting of amino acids 3-8 and 1-4 of the human Aβ peptide sequence, respectively. The epitope for the 3B6 and 3B6 biotin antibodies is a sequence consisting of 119-140 of the α-synuclein protein sequence.

Example 2: Preparation of 6E10 plate 30 μg of 6E10 antibody (anti-Aβ protein, BioLegend) is diluted with 10 ml of coating buffer (sigma), 100 μl is dispensed into each well on a plate (Nunc), and then in a refrigerator at 4 ° C. Reacted for a day. The plate was washed 3 times with PBS and D.I. After dispensing 240 μl of blocking buffer in which 1% Block Ace was dissolved in W, the reaction was carried out at room temperature for 2 hours. The plate was washed 3 times with PBS, dried at room temperature for 30 minutes and then used.

Example 3: Preparation of 3B6 plate 20 μg of 3B6 antibody (anti-α-synuclein protein, Novus Biologicals) is diluted with 10 ml of coating buffer (sigma), 100 μl is dispensed into each well on a plate (Nunc), and then at 4 ° C. Reacted in the refrigerator for a day. The plate was washed 3 times with PBS and D.I. After dispensing 240 μl of blocking buffer in which 1% Block Ace was dissolved in W, the reaction was carried out at room temperature for 2 hours. The plate was washed 3 times with PBS, dried at room temperature for 30 minutes and then used.

Example 4: Preparation of control group For the positive control group, 990 μl of PBST was added to 10 μl of recombinant Aβ1-42 (rec.Aβ) (1 μg / ml), and 100 μl was used. For the positive control group, 10 μl of recombinant α-synuclein (1 mg / ml) was added with 990 μl of PBST, and 100 μl was used. As a negative control group (negative control), 100 μl of PBS was used.

Example 5: Preparation of samples Samples were prepared based on two samples. Frozen plasma samples were thawed in a 37 ° C. heat block for 15 minutes and then used after vortexing for 30 seconds. rec. Samples spiked with 1 ng of Aβ1-42 were 20 μl of plasma with 8.08 μl of HBR1 (0.123 mg / ml) and 180 μl of PBST, rec. 20 μl of Aβ1-42 (1 ng / 10 μl) was mixed and prepared to make a total of 228.08 μl. rec. Samples spiked with 1 ng of Aβ9-42 biotin were plasma 20 μl with HBR1 (0.123 mg / ml) 8.08 μl and PBST 180 μl, rec. 20 μl of Aβ9-42 biotin (1 ng / 10 μl) was mixed and prepared to make a total of 228.08 μl. rec. Samples spiked with Aβ9-42 1 ng were plasma 20 μl with HBR1 (0.123 mg / ml) 8.08 μl and PBST 180 μl, rec. 20 μl of Aβ9-42 (1 ng / 10 μl) was mixed and prepared to make a total of 228.08 μl. For the sample in which 1 μg of recombinant α-synuclein was spiked, 8.08 μl of HBR1 (0.123 mg / ml), 180 μl of PBST, and 20 μl of recombinant α-synuclein (1 μg / 10 μl) were mixed with 20 μl of plasma, and the total amount was 228. It was prepared to be 08 μl. In addition, the sample in which the recombinant peptide was not spiked was prepared by mixing 8.08 μl of HBR1 (0.123 mg / ml) and 200 μl of PBST in 20 μl of plasma to make a total of 228.08 μl.

Example 6: Incubation
In Example 5, rec. Samples prepared by treating Aβ1-42 were incubated in an incubator at 37 ° C. for 4 days and 6 days, respectively. In Example 5, rec. Samples prepared by treating Aβ1-42 were incubated in a 37 ° C. incubator for 2 days, 3 days, 4 days, and 5 days, respectively. rec. Samples untreated with Aβ1-42 were incubated in a 37 ° C. incubator for 0 days and 5 days each. Then, in the fifth embodiment, rec. Samples prepared by treating Aβ9-42 biotin were incubated for 6 days. In Example 5, rec. Samples prepared by treating Aβ9-42 were incubated for 2 days, 3 days and 4 days, respectively. In addition, the samples prepared by treating the recombinant α-synuclein in Example 5 were incubated for 0 days, 2 days, 4 days, and 6 days, respectively.

Example 7: rec. Detection of Aβ oligomers using MDS (multimer detection system) from samples incubated for 4 and 6 days after treatment with Aβ1-42 6E10 coated plate (3 μg / ml), positive control group, negative Control group and rec. Samples treated with Aβ1-42 and incubated for 4 days and 6 days were dispensed by 100 μl each and then reacted at room temperature for 1 hour. The plate was washed 3 times with TBST, and the FF51-HRP antibody was diluted 1/1000 in buffer A and dispensed 100 μl each, and then reacted at room temperature for 1 hour. After washing the plate 3 times with TBST, 100 μl of ECL solution was dispensed. The plate that reacted with the ECL was inserted into a perkinelmer and the luminescence signal was measured. The result is shown in FIG. 1a below.

1a and 1b show rec. It is shown that after the addition of Aβ1-42, the signal of the AD sample is increased as compared with the signal of the Non AD sample by the incubation time. The difference between AD and Non AD is shown for each condition incubated for 4 and 6 days.

As seen from FIGS. 1a and 1b, the signal of Aβ oligomer in the sample of AD patient is higher when compared with the sample of Non AD patient. In the sample of AD patient, the defense system that suppresses the formation of Aβ oligomer is higher in the non AD patient. It is judged that this is because it is not activated.

Example 8: rec. Detection of Aβ oligomers using MDS (multimer detection system) from samples incubated for 2 days, 3 days, 4 days, and 5 days after treatment with Aβ1-42 6E10 coating plate (3 μg / ml) In addition, positive control group, negative control group and rec. 100 μl of each sample treated with Aβ1-42 and incubated for 2 days, 3 days, 4 days, and 5 days was dispensed, and then reacted in a stationary state in a 27 ° C. incubator for 1 hour. The plate was washed 3 times with TBST, and 100 μl of the FF51-HRP antibody was dispensed into buffer A at 10 ng / ml. The plate was reacted in a 27 ° C. incubator in a stationary state for 1 hour, washed 3 times with TBST, and then 100 μl of ECL solution was dispensed. The plate that reacted with the ECL was inserted into a perkinelmer and the luminescence signal was measured. The result is shown in FIG. 1c below.

FIG. 1c shows rec. After the addition of Aβ1-42, the signal of the AD sample was 1.15 times, 1.34 times, 1.65 times and 1 of the signal of the Non AD sample depending on the incubation time of 2, 3, 4, and 5 days. It shows that it has increased by .84 times. It shows that as the number of incubation days increases, the difference between AD and Non AD becomes wider and wider.

As seen from FIG. 1c, when compared with the Non AD patient sample, the signal of Aβ oligomer in the AD patient sample was higher because the defense system that suppresses the formation of Aβ oligomer in the AD patient sample was more active in the Non AD patient sample. It is judged that this is because it does not change.

Example 9: rec. Treated with Aβ1-42 for 5 days, rec. Detection of Aβ oligomer (oligomer) using MDS (multimer detection system) from samples incubated for 0 days and 5 days without treatment of Aβ1-42. Positive control group, negative on 6E10 coating plate (3 μg / ml) Control group and rec. Treated with Aβ1-42 for 5 days, rec. 100 μl of each sample incubated for 5 days for 0 days without treatment of Aβ1-42 was dispensed, and then reacted in a stationary state in a 27 ° C. incubator for 1 hour. The plate was washed 3 times with TBST, and 100 μl of the FF51-HRP antibody was dispensed into buffer A at 10 ng / ml. The plate was reacted in a 27 ° C. incubator in a stationary state for 1 hour, washed 3 times with TBST, and then 100 μl of ECL solution was dispensed. The plate that reacted with the ECL was inserted into a perkinelmer and the luminescence signal was measured. The result is shown in FIG. 1d below.

FIG. 1d shows rec. Incubation for 5 days after addition of Aβ1-42, rec. Data obtained by measuring Aβ oligomer from a sample incubated for 0 days and 5 days without adding Aβ1-42. Signals of AD sample with time when Aβ1-42 was spiked and when it was not spiked. And the increased state of the signal of the Non AD sample, rec. Only samples incubated for 5 days after the addition of Aβ1-42 show a difference between AD and Non AD.

From FIG. 1d, it was judged that the high signal of Aβ oligomer from the sample of AD patient is because the defense system that suppresses the formation of Aβ oligomer is not activated as much as that of Non AD patient in the sample of AD patient. Therefore, when Aβ1-42 is spiked, it is judged to have an effect on the defense system that suppresses the formation of Aβ oligomers.

Example 10: rec. Detection of Aβ oligomers using MDS (multimer detection system) from samples incubated for 6 days after treatment with Aβ9-42 biotin 6E10 coated plates (3 μg / ml) were subjected to positive control group, negative control group, rec. 100 μl of each sample treated with Aβ9-42 biotin and incubated for 6 days was dispensed and then reacted at room temperature for 1 hour. The plate was washed 3 times with TBST, the FF51-HRP antibody was diluted 1/1000 in buffer A, 100 μl of each was dispensed, and then the reaction was carried out at room temperature for 1 hour. After washing the plate 3 times with TBST, 100 μl of ECL solution was dispensed. The plate that reacted with ECL was inserted into a luminometer device (perkinelmer), the luminescence signal was measured, and the results are summarized in FIG. 2a below.

FIG. 2a shows rec. Binding to Aβ. It shows that when Aβ9-42 biotin is spiked and incubated for 6 days, the signal in the AD patient group is higher than that in the normal group.

Seen from FIG. 2a, rec. It was determined that Aβ9-42 biotin binds to Aβ and promotes agglutination, and is classified by changes in the quantitative and magnitude portions of the antigen.

Example 11: rec. Detection of Aβ oligomers using MDS (multimer detection system) from samples incubated for 2 days, 3 days, and 4 days after treatment with Aβ9-42 biotin 6E10 coated plate (3 μg / ml), positive control group, Negative control group, rec. 100 μl of each sample treated with Aβ9-42 and incubated for 2 days, 3 days and 4 days was dispensed and then reacted in a stationary state in a 27 ° C. incubator for 1 hour. The plate was washed 3 times with TBST, and FF51-HRP antibody was produced in buffer A so as to have a concentration of 10 ng / ml, and then 100 μl was dispensed. The plate was reacted in a 27 ° C. incubator in a stationary state for 1 hour, washed 3 times with TBST, and then 100 μl of ECL solution was dispensed. The plate that reacted with ECL was inserted into a luminometer device (perkinelmer), the luminescence signal was measured, and the results are summarized in FIG. 2b below.

FIG. 2b shows rec. Binding to Aβ. When Aβ9-42 was spiked and incubated for 2, 3, and 4 days, the signal of the AD patient group increased 1.1 times, 1.52 times, and 1.84 times that of the normal group over time. Show that.

Seen from FIG. 2b, rec. It was determined that Aβ9-42 biotin binds to Aβ and promotes agglutination, and is distinguished by changes in the quantitative and sized portions of the antigen.

Example 12: Detection of α-synuclein oligomer using MDS (multimer detection system) from a sample incubated for 0 days, 2 days, 4 days, and 6 days after treating recombinant α-synuclein 3B6 coating plate ( 100 μl of each of the samples treated with the positive control group, the negative control group, and the recombinant α-synuclein and incubated for 0 days, 2 days, 4 days, and 6 days was dispensed into 2 μg / ml), and then in the 27 ° C. incubator. Reacted for 1 hour in a stationary state. The plate was washed 3 times with TBST, 3B6-biotin antibody was produced in buffer A at 2 μg / ml, and then 100 μl was dispensed. The plate was reacted in a 27 ° C. incubator in a stationary state for 1 hour and washed 3 times with TBST. Streptavidin-HRP was diluted 1/5000 times in buffer A, dispensed in 100 μl portions, and then the plate was reacted in a standing state in a 27 ° C. incubator for 1 hour and washed 3 times with TBST. 100 μl of the ECL solution was dispensed. The plate that reacted with ECL was inserted into a luminometer device (perkinelmer), the luminescence signal was measured, and the results are summarized in FIG. 3 below.

FIG. 3 is data showing changes in the signal of the PD sample and the signal of the Non PD sample over time for 0 days, 2 days, 4 days, and 6 days of incubation after the addition of the recombinant α-synuclein. At day 0, the signal ratio of the PD sample was 0.92 times that of the Non PD signal, but during incubation for 2 days, 4 days, and 6 days, 1.34 times, 1.76 times, 1 It was shown that it increased by .78 times.

As seen from FIG. 3, when compared with the sample of Non PD patient, the signal of α-synuclein was high in the sample of PD patient because the defense system that suppresses the formation of α-synuclein oligomer in the sample of PD patient is Non. It is judged that this is because it is not as active as PD patients.

Although the specific parts of the present invention have been described in detail above, such a specific description is merely a desirable embodiment for a person having ordinary knowledge in the art, and the scope of the present invention is limited thereto. It is clear that it is not limited. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (26)

(A) The step of spike the monomer type or multimer type of the aggregated-formed polypeptide on the blood sample to be analyzed, and
(B) A step of incubating the result of the step (a) to additionally form an aggregated form of the aggregated-forming polypeptide, and
(C) A step of contacting the result of the step (b) with a binder-label in which a signal generating label is bound to a binder that binds to the aggregated form of the aggregated polypeptide.
(D) A step of detecting a signal generated from the aggregate-binding agent-label bound to the aggregated form of the formed polypeptide, and
Including
Incubation of the step (b), the spiked aggregated form - forming monomeric or multimeric forms of the polypeptide is carried out enough time can multimerization by the blood sample, aggregated form in a blood sample - aggregation of forming polypeptide How to detect the shape. The blood sample that multimerizes the monomeric or multimer type of the spiked aggregate-forming polypeptide is a human blood sample having a disease associated with the multimer type of the aggregate-formed polypeptide. The method according to claim 1. Wherein enough incubation time can multimerized by the blood sample, aggregated form - the signal generated by using human blood samples having a disease involved the multimeric forms of the forming polypeptide, utilizing a normal human blood sample The method according to claim 2, wherein the time is sufficient to make the signal 1.5 to 20 times larger than the signal generated. The method according to claim 1, wherein the blood sample is plasma. The aggregated-forming polypeptide includes Aβ peptide, tau protein, prion, α-synuclein, Ig light chain, serum amyloid A, transsiletin, cystatin C, β2-microglobulin, huntingtin, superoxide dismutase, serpine and The method of claim 1, wherein the method was selected from the group consisting of amylin. The method of claim 5, wherein the aggregated-forming polypeptide is an Aβ peptide, tau protein or α-synuclein. The method according to claim 1, wherein the monomer type of the aggregated-formed polypeptide is an Aβ peptide consisting of the amino acid sequence of SEQ ID NO: 1 or α-synuclein consisting of the amino acid sequence of SEQ ID NO: 2. The method according to claim 1, wherein a buffer solution is further added to the product of the step (a). The method according to claim 8, wherein the buffer solution is added in an amount of 3 to 15 times (v / v) with respect to the blood sample . The method according to claim 8, wherein the buffer solution is a phosphate buffer solution containing a nonionic surfactant. The additional formation of the aggregated form of the aggregated polypeptide of the step (b) is characterized by incubating the result of the step (a) at a temperature of 1 to 50 ° C., claim 1. The method described in. The aggregated form of the step (b) -the additional formation of the aggregated form of the polypeptide is carried out by incubating the result of the step (a) for 1 to 12 days, according to claim 1. The method described. The steps (c) and (d) are
(C-1) A step of contacting the result of the step (b) with a capture antibody that recognizes an epitope on the aggregate-forming polypeptide that captures the aggregate.
(C-2) A step of contacting the captured aggregate with a detection antibody that recognizes an epitope on the aggregate-forming polypeptide.
(C-3) At the stage of detecting the aggregate-detection antibody complex,
The method according to claim 1, wherein the method is performed by a method including. 13. The method of claim 13, wherein the detection antibody is a detection antibody that recognizes an epitope that is the same as or overlaps with the epitope of step (c-1). 13. The method of claim 13, wherein the captured antibody is bound to a solid substrate. 13. The method of claim 13, wherein the detection antibody has a label that produces a detectable signal. The method according to claim 16, wherein the label bound to the detection antibody is a compound label, an enzyme label, a radioactivity label, a fluorescent label, a luminescent label, a chemiluminescent label or a FRET label. A kit for detecting agglutinating-forming polypeptides in blood samples .
Aggregate-Forming polypeptides in monomeric or multimer form,
Includes a binder-label in which a signaling label is attached to a binder that binds to the aggregate-forming polypeptide.
The monomer type of the aggregated-formed polypeptide is an Aβ peptide consisting of the amino acid sequence of SEQ ID NO: 1.
A kit for detecting the aggregated form of an aggregated-formed polypeptide in a blood sample by detecting a signal generated from the binder-label bound to the aggregated form of the aggregated-forming polypeptide. The kit according to claim 18, wherein the blood sample is plasma. The kit according to claim 18, wherein the kit further contains a buffer solution. The kit according to claim 20, wherein the buffer solution is a phosphate buffer solution containing a nonionic surfactant. The kit according to claim 18, wherein the kit further comprises a capture antibody that recognizes an epitope on an aggregate-forming polypeptide and a detection antibody that recognizes the epitope recognized by the capture antibody. .. 22. The kit according to claim 22, wherein the detection antibody is a detection antibody that recognizes an epitope that is the same as or overlaps with the epitope recognized by the capture antibody. 22. The kit of claim 22, wherein the captured antibody is bound to a solid substrate. 22. The kit of claim 22, wherein the detection antibody has a label that produces a detectable signal. The kit according to claim 25, wherein the label bound to the detection antibody is a compound label, an enzyme label, a radioactivity label, a fluorescent label, a luminescent label, a chemiluminescent label or a FRET label.